Regulation of IKKbeta by miR-199a affects NF-kappaB activity in ovarian cancer cells

Abstract

Cancer progression is an abnormal form of tissue repair characterized by chronic inflammation. IkappaB kinase-beta (IKKbeta) required for nuclear factor-kappaB (NF-kappaB) activation plays a critical role in this process. Using EOC cells isolated from malignant ovarian cancer ascites and solid tumors, we identified IKKbeta as a major factor promoting a functional TLR-MyD88-NF-kappaB pathway that confers to EOC cell the capacity to constitutively secrete proinflammatory/protumor cytokines and therefore promoting tumor progression and chemoresistance. Furthermore, we describe for the first time the identification of the microRNA hsa-miR-199a as a regulator of IKKbeta expression. Our study describes the property of ovarian cancer cells to enhance the inflammatory microenvironment as a result of the expression of an active IKKbeta pathway. Identification of these markers in patients' tumor samples may facilitate the adequate selection of treatment and open new venues for the development of effective therapy for chemoresistant ovarian cancers.

Figure 1

Differential expression of cytokines between Type I and II epithelial ovarian cancer (EOC) cells. Cytokine production was determined in the supernatant from EOC cells. Type I EOC cells expresses significant levels of inflammatory cytokines. No inflammatory cytokines were detected in Type II EOC cells in any condition. Representative figure of 20 evaluated EOC cells. Each sample was performed in triplicates.

Figure 2

Determination of nuclear factor-κB (NF-κB) activity in ovarian cancer cells transfected with a luciferase reporter construct containing two κB-binding sites. (a) Endogenous cyclic NF-κB activity was observed in Type I but not Type II epithelial ovarian cancer (EOC) cells during a period of 12 h. (b) Treatment with lipopolysaccharide (LPS) and paclitaxel induced NF-κB activity in Type I but not in Type II EOC cells, as represented by data at 12 h after treatment. *P<0.05. (c) Treatment of Type I EOC cells with 2 μM EriB, a NF-κB inhibitor, blocks the constitutive cytokine production, indicating its NF-κB dependence. EriB, Eriocalyxin B. *P<0.05. Representative experiment of two Type I and II EOC cell lines. Similar results were observed with other cells of the same type. n=3 per sample per time point.

Figure 3

Differential expression of IκBα and IKK subunits between Type I and II epithelial ovarian cancer (EOC) cells. (a) Western blot analysis for IκBα expression in ovarian cancer cells. Note the lack of IκBα expression in Type I EOC cells compared to Type II. Low IκBα expression corresponds to high MyD88 expression levels. (b) Differential expression of IKKα and IKKβ in Type I and II EOC cells. Type I EOC cells are characterized by a high IKKβ/IKKα ratio. Representative figure of four Type I and five Type II EOC cells out of 40 cancers evaluated in five independent experiments. Each experiment was performed in triplicates.

Figure 4

Effect of ectopic overexpression of IKKβ in Type II epithelial ovarian cancer (EOC) cells. (a) Transient overexpression of a constitutively active form of IKKβ (IKKβ S177E S181E) in Type II EOC cells induced a significant decrease in IκBα expression and increase in MyD88 expression. No change in IKKα expression was observed. (b) Overexpression of IKKβ S177E S181E in Type II EOC cells promotes cytokine production. (c) Transient overexpression of WT IKKβ in a Type II MyD88-positive stable transfectant cell line restored the functionality of the TLR4 pathway, as determined by lipopolysaccharide (LPS)-induced cytokine production. *P<0.05. pCMV2-IKKEE, plasmid expressing a constitutively active form of IKKβ (IKKβ S177E S181E). MOCK, mock transfection with the empty plasmid pCMV2. Representative figure of an experiment using A2780 cells. Similar results were obtained with two additional Type II EOC cells. (d) Differential effect of TNF-α on ovarian cancer cells. Treatment with TNF-α, 100 ng/ml, (24 h) induce cell death in Type II but not in Type I ovarian cancer cells. Six representative cell lines NT, nontreatment. (e) Induction of caspase activity by TNF-α 100 ng/ml, (24 h) in Type II EOC cells. Representative experiment of at least eight cell lines. Each experiment was performed in triplicates. *P<0.0001. (f) TNF-α treatment induces the phosphorylation of IKKβ but not IKKα in Type I EOC cells. No changes are observed in Type II cells. Representative experiment performed with five Type I and five Type II cell lines.

Figure 4

Effect of ectopic overexpression of IKKβ in Type II epithelial ovarian cancer (EOC) cells. (a) Transient overexpression of a constitutively active form of IKKβ (IKKβ S177E S181E) in Type II EOC cells induced a significant decrease in IκBα expression and increase in MyD88 expression. No change in IKKα expression was observed. (b) Overexpression of IKKβ S177E S181E in Type II EOC cells promotes cytokine production. (c) Transient overexpression of WT IKKβ in a Type II MyD88-positive stable transfectant cell line restored the functionality of the TLR4 pathway, as determined by lipopolysaccharide (LPS)-induced cytokine production. *P<0.05. pCMV2-IKKEE, plasmid expressing a constitutively active form of IKKβ (IKKβ S177E S181E). MOCK, mock transfection with the empty plasmid pCMV2. Representative figure of an experiment using A2780 cells. Similar results were obtained with two additional Type II EOC cells. (d) Differential effect of TNF-α on ovarian cancer cells. Treatment with TNF-α, 100 ng/ml, (24 h) induce cell death in Type II but not in Type I ovarian cancer cells. Six representative cell lines NT, nontreatment. (e) Induction of caspase activity by TNF-α 100 ng/ml, (24 h) in Type II EOC cells. Representative experiment of at least eight cell lines. Each experiment was performed in triplicates. *P<0.0001. (f) TNF-α treatment induces the phosphorylation of IKKβ but not IKKα in Type I EOC cells. No changes are observed in Type II cells. Representative experiment performed with five Type I and five Type II cell lines.

Figure 5

MicroRNAs (miRNA) profiles in Type I and II epithelial ovarian cancer (EOC) cells, and their relationship to IKKβ expression. (a) Type I and II cells have similar levels of IKKβ mRNA, as determined by reverse transcription (RT)–PCR. (b) miRNA profiling of one Type I and two Type II cell lines by Invitrogen NCODE miRNA microarray. (c) Panel of differentially expressed miRNA in Type I and II cells. Note the similarity of miRNA expression between the Type II cell lines and their similar differences in relation to Type I. Red indicates miRNA upregulated in Type II versus Type I; green indicates miRNA downregulated in Type II versus Type I. (d) Three putative hsa-miR-199a-binding sites within the 3′-UTR region of the IKKB mRNA, as predicted by Pictar (http://www.pictar.bio.nyu.edu/, and the algorithm ‘PicTar predictions in vertebrates, flies and nematodes’ was selected). Red sequences, hsa-miR-199a; black sequences, putative binding sites on the 3′-UTR of IKKB mRNA. (e) qRT–PCR quantification of hsa-miR-199a in ovarian cancer cells. Data normalized to β-2 macroglobulin. Note the high expression levels of hsa-miR-199a in Type II cells compared to Type I cells.

Figure 6

Regulation of IKKβ expression by hsa-miR-199a. (A) (a) Transient transfection of Type I cells with Pre-miR-199a inhibits IKKβ expression. (b) Transient transfection of Type II cells with anti-miR-199a induced IKKβ expression. (B) miR-199a suppressed the IKKB 3′-UTR Luciferase reporter activity compared to mock transfection, whereas the negative control miRNA (miR-NC no. 1) did not result in any changes. Diagram of the Luciferase reporter plasmid to study the function of the 3′-UTR of IKKB mRNA. The reporter consists of a Luciferase gene with the IKKB 3′-UTR driven by a cytomegalovirus promoter. *P<0.001.

Figure 7

Model of Type I and II EOC cells. Type I epithelial ovarian cancer (EOC) cells have high levels of IKKβ expression due to low hsa-miR-199a; therefore, when stimulated, nuclear factor-κB (NF-κB) activation leads to cytokine production, cell proliferation and induction of antiapoptotic proteins. In Type II cells expression of IKKβ is low due to high hsa-miR-199a expression levels, therefore an incomplete TLR4–MyD88–NF-κB pathway cannot respond to ligands, resulting in no cytokine production and chemosensitivity.